RESUMEN
Lowicryl resins enable processing of biological material for electron microscopy at the lowest temperatures compatible with resin embedding. When combined with high-pressure freezing and freeze-substitution, Lowicryl embedding supports preservation of fine structural details and fluorescent markers. Here, we analysed the applicability of Lowicryl HM20 embedding for focused ion beam (FIB) scanning electron microscopy (SEM) tomography of Drosophila melanogaster embryonic and larval model systems. We show that the freeze-substitution with per-mill concentrations of uranyl acetate provided sufficient contrast and an image quality of SEM imaging in the range of similar samples analysed by transmission electron microscopy (TEM). Preservation of genetically encoded fluorescent proteins allowed correlative localization of regions of interest (ROI) within the embedded tissue block. TEM on sections cut from the block face enabled evaluation of structural preservation to allow ROI ranking and thus targeted, time-efficient FIB-SEM tomography data collection. The versatility of Lowicryl embedding opens new perspectives for designing hybrid SEM-TEM workflows to comprehensively analyse biological structures. LAY DESCRIPTION: Focused ion beam scanning electron microscopy is becoming a widely used technique for the three-dimensional analysis of biological samples at fine structural details beyond levels feasible for light microscopy. To withstand the abrasion of material by the ion beam and the imaging by the scanning electron beam, biological samples have to be embedded into resins, most commonly these are very dense epoxy-based plastics. However, dense resins generate electron scattering which interferes with the signal from the biological specimen. Furthermore, to improve the imaging contrast, epoxy embedding requires chemical treatments with e.g. heavy metals, which deteriorate the ultrastructure of the biological specimen. In this study we explored the applicability of an electron lucent resin, Lowicryl HM 20, for focused ion beam scanning electron microscopy. The Lowicryl embedding workflow operates at milder chemical treatments and lower temperatures, thus preserving the sub-cellular and sub-organellar organization, as well as fluorescent markers visible by light microscopy. Here we show that focus ion beam scanning electron microscopy of Lowicryl-embedded fruit flies tissues provides reliable imaging revealing fine structural details. Our workflow benefited from use of transmission electron microscopy for the quality control of the ultrastructural preservation and fluorescent light microscopy for localization of regions of interest. The versatility of Lowicryl embedding opens up new perspectives for designing hybrid workflows combining fluorescent light, scanning, and transmission electron microscopy techniques to comprehensively analyze biological structures.
Asunto(s)
Resinas Acrílicas , Drosophila melanogaster/embriología , Técnicas Histológicas/métodos , Microscopía Electrónica de Rastreo/métodos , Adhesión del Tejido , Animales , Substitución por Congelación , Congelación , Microscopía Electrónica de Transmisión/métodosRESUMEN
The frequency of ADHD in the aging population and its relationship to late-life cognitive decline has not been studied previously. To address this gap in our understanding, the Wender-Utah ADHD Rating scale (WURS) was administered to 310 geriatric subjects with cognitive status ranging from normal cognition to mild cognitive impairment to overt dementia. The frequency of WURS-positive ADHD in this sample was 4.4%. WURS scores were not related to cognitive diagnoses, but did show nonlinear associations with tasks requiring sustained attention. The frequency of ADHD appears stable across generations and does not appear to be associated with MCI or dementia diagnoses. The association of attentional processing deficits and WURS scores in geriatric subjects could suggest that such traits remain stable throughout life. Caution should be considered when interpreting cognitive test profiles in the aging population that exhibit signs and symptoms of ADHD, as attentional deficits may not necessarily imply the existence of an underlying neurodegenerative disease state.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Coronavirus Humano 229E/enzimología , Guanosina Trifosfato/metabolismo , ARN Helicasas/metabolismo , Animales , Baculoviridae/genética , Secuencia de Bases , Células Cultivadas , Coronavirus Humano 229E/genética , Humanos , Insectos , Datos de Secuencia Molecular , Nucleósido-Trifosfatasa , ARN Helicasas/genética , Recombinación GenéticaRESUMEN
The arterivirus equine arteritis virus nonstructural protein 10 (nsp10) has previously been predicted to contain a Zn finger structure linked to a superfamily 1 (SF1) helicase domain. A recombinant form of nsp10, MBP-nsp10, was produced in Escherichia coli as a fusion protein with the maltose-binding protein. The protein was partially purified by affinity chromatography and shown to have ATPase activity that was strongly stimulated by poly(dT), poly(U), and poly(dA) but not by poly(G). The protein also had both RNA and DNA duplex-unwinding activities that required the presence of 5' single-stranded regions on the partial-duplex substrates, indicating a 5'-to-3' polarity in the unwinding reaction. Results of this study suggest a close functional relationship between the arterivirus nsp10 and the coronavirus helicase, for which NTPase and duplex-unwinding activities were recently demonstrated. In a number of biochemical properties, both arterivirus and coronavirus SF1 helicases differ significantly from the previously characterized RNA virus SF1 and SF2 enzymes. Thus, the combined data strongly support the idea that nidovirus helicases may represent a separate group of RNA virus-encoded helicases with distinct properties.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Arterivirus/enzimología , Coronavirus/enzimología , ADN Helicasas/fisiología , Proteínas de Escherichia coli , Proteínas de Transporte de Monosacáridos , ARN Helicasas/fisiología , Proteínas no Estructurales Virales/fisiología , Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , ADN Viral/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión a Maltosa , ARN Viral/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Cloruro de Sodio/farmacología , Dedos de ZincRESUMEN
The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.
Asunto(s)
Coronavirus Humano 229E , Coronavirus/enzimología , ADN Helicasas/genética , ADN Helicasas/metabolismo , Expresión Génica , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas Virales , Adenosina Trifosfatasas/metabolismo , Animales , Baculoviridae/metabolismo , Secuencia de Bases , Cromatografía de Afinidad , ADN/metabolismo , ADN Helicasas/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Histidina/metabolismo , Insectos/metabolismo , Datos de Secuencia Molecular , Mutación Puntual , Polinucleótidos/farmacología , ARN Helicasas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas no Estructurales ViralesRESUMEN
Coronavirus gene expression involves proteolytic processing of the gene 1-encoded polyproteins and a key enzyme in this process is the virus-encoded 3C-like proteinase. In this study, we describe the biosynthesis of the human coronavirus 229E 3C-like proteinase in Escherichia coli and the substrate specificity of the purified protein. Using immunofluorescence microscopy, we have also investigated the subcellular localization of the 3C-like proteinase and have found a punctate, perinuclear distribution of the proteinase in virus-infected cells.
Asunto(s)
Coronavirus Humano 229E , Coronavirus/enzimología , Cisteína Endopeptidasas/metabolismo , Proteínas Virales , Proteasas Virales 3C , Secuencia de Aminoácidos , Línea Celular , Cisteína Endopeptidasas/genética , Humanos , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
The replication of coronaviruses involves proteolytic processing of the gene 1 translation products, pp1a and pp1ab. One of the key enzymes in this process is predicted to be a virus-encoded 3C-like proteinase. In this report, we describe a bacterial system that has allowed us to express and characterize a recombinant murine coronavirus (MHV-JHM) 3C-like proteinase. The partially purified protein has been shown to exhibit proteolytic activity in trans and mutation analysis has been used to demonstrate the indispensability of Cys-3495 for enzymatic activity. Finally, the effect of class-specific proteinase inhibitors on the trans cleavage activity of the MHV 3C-like proteinase has been used to demonstrate the functional and structural homology of this enzyme to the picornavirus 3C proteinases.